287
J . Med. Chem. 1986,29, 287-291
Saturated Analogues of Poison Ivy Allergens. Synthesis of trans ,trans - and cis ,trans -3-Alkyl-1,2-cyclohexanediolsand Sensitizing Properties in Allergic Contact Dermatitis Jean-Pierre Lepoittevin and Claude Benezra* PIREN "SantB et Environnement", Laboratoire de Dermato-Chimie, Associe au CNRS ( U A 31), Uniuersite Louis Pasteur, Clinique Dermatologique, CHU, 67091 Strasbourg, France. Received June 19, 1985 Saturated analogues of poison ivy and oak allergens (3-alkylcatechols), i.e. trans,trans-3-alkyl-1,2-cyclohexanediols (alkyl = CH8, n-C5H11,n-CloH21,n-C15H3J,have been prepared and used to sensitize guinea pigs. Only long-chain derivatives (carbon chain length > Clo) are contact sensitizers. The sensitized animals cross-react to PDC (i.e. pentadecylcatechol, one of the allergens of poison ivy), but the converse is not true (PDC-sensitized animals do not react to cyclohexanediols). cis,trans-3-n-Pentadecyl-l,2-cyclohexanediol has also been synthesized and shown to be a sensitizer. There is not cross-reaction between trans,trans- and cis,trans-3-n-pentadecylcyclohexanediols,excluding a common skin metabolite.
Allergic contact dermatitis to toxicodendron (poison ivy, oak, sumac) is a major cause of morbidity in the United States (80% of the U.S. population is allergic to them'). The allergenic principles contained in the resin of these plants, called urushiol, are a group of catechols2 differing mainly in the length and degree of unsaturation of the 3-n-alk(en)ylside chain. In the last years, several attempts have been made to induce immune tolerance to these compounds in the guinea pig, in particular by the intravenous injection of urushiol derivative^,^ 3-n-pentadecylcatechol coupled to autologous blood cells: or incorporated into l i p ~ s o m e . However, ~ since urushiols are highly toxic and potent primary irritants, their use for inducing tolerance in nonsensitive humans is heavily compromised. It would therefore be useful to make nontoxic analogues of these products, another approach to tolerance being the use of modified related substances, i.e. poor sensitizers. Thus sodium dinitrobenzenesulfonate6 or dinitrothiocyanobenzene' induce tolerance to dinitrofluorobenzene. It is generally admitted that low-molecular-weight compounds are not immunogenic unless covalently attached to proteims Unfortunately, high chemical reactivity of allergens often accompanies their toxicity, as is the case, for instance, for alkylcatechols. An exception to the rule that immunogenic conjugates may only be formed by covalent binding appears to be the example of some lowmolecular-weight compounds strongly bound to cell membranes, This is the case for some metal salts? or picric acid, a low-molecular-weightsensitizer, which is an example of a compound that, although not covalently associating with carriers, gives rise to an immunogenic complex.1° Experiments using methylated albumins or electrostat(1) Epstein, W. L. Cutis 1974, 13, 544-548. (2) Billet, S.; Craig, J. C.; Carbett, M. D.; Wickery, J. F. Phytochemistry 1976, 15, 533-535. (3) Watson, E. S.; Murphy, J. C.; Wirth, P. W.; Waller, C. W.; Elsohly, M. A. J. Invest. Dermatol. 1981, 76, 164-170. (4) Watson, E. S.; Murphy, J. C.; Wirth, P. W.; Elsohly, M. A,; Skierkowski, P. J. Pharm. Sci. 1981, 70, 785-789. (5) Colby, S. L.; Artis, W. M.; Rietschel, R. L. J.Invest. Dermatol. 1983,80, 145-149. ( 6 ) Frey, J . R.; De Weck, A. L.; Geleick, H. J. Invest. Derrnatol.
1964, 42, 189-196. (7) Iijima, M.; Katz, S. I. J. Invest. Dermatol. 1983,81, 325-330. (8) Dupuis, G.; Benezra, C. "Allergic contact Dermatitis to Simple Marcel Dekker: New Chemicals: A Molecular ADDroach": -_ York, 1982. (9) . , Hutchinson. F.: McLeod. T. M.: Raffler. E. J. Br. J.Dermatol. 1975, 93, 557. Polak, L.f Frey,J. R. Znt. Arch. Allergy Appl. Immunol. 1973, 44, 51-61. (10) Chase, M. W.; Maguire, M. C. Znt. Arch. Allergy Appl. Immunol. 1973, 45, 513-542.
Scheme I"
Rd 1
2
U
R 3a-d
" (a) R,CuLi, ( b ) ClSiMe,, (c) BH,-Me$, ( d ) H,O,, NaOH. For 3: 3a, R = CH, ; 3b, R = n-C,H,, ; 3c, R = n-C,,H,,; 3d, R = n-ClsH,,. Scheme I1
ically charged polypeptides as carriers indicate, however, that molecules strongly attached to such carriers by noncovalent bonds may also behave as immunogens." Urushiol is an extremely lipophilic hapten and concentrates readily in cell membranes. It was shown to be highly soluble in but not covalently bound to the cell membrane, since, although it was not removed from the membrane by aqueous extraction, it could nevertheless be extracted with Me2S0.12 Urushiol-specific blastogenesis can be elicited from cultured peripheral blood lymphocytes by noncovalently bound hapten associated with the membranes because of ita lipophilic character.13 Finally Byers et d. have shown that insertion of lipophilic molecules into the lipid phase of tumor cells can induce a modification of tumor imm~nogenicity.'~ It therefore seemed of interest to synthesize nonelectrophilic and nontoxic compounds capable of forming strong noncovalent bonds. This paper reports the preparation of such compounds, alkylcyclohexanediols, saturated analogues of poison ivy and poison oak pentadecylcatechols, as well as a study of their sensitizing power in guinea pigs. (11) Plescia, 0. J.; Braun, W.; Imperato, S.; Cora-Block, E.; Joras-
kova, L.; Schimbor, C. "Nucleic Acids in Immunology"; Springer: New York, 1968. (12) Watson, E. S.; Murphy, J. C.; Wirth, P. W.; Elsohly, M. A.; Skierkowski, P. J. Pharm. Sci. 1981, 70, 785-789. (13) Byers, V. S.; Epstein, W. L.; Castagnoli, N.; Baer, H. J. Clin. Invest. 1979, 64, 1437-1448. (14) Byers, V. S.; Baldwin, R. W. Symp. Mol. Cell. Biol. 1979, 603-622.
0022-2623/86/1829-0287$01.50/00 1986 American Chemical Society
288 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2 Table I. Characteristic NMR Data
'H NMRb, ppm R, trans,trans series 3a, CH3 3b, n-C5Hl, 3c, n-CioHzi 3 4 n-Ci5H31
coupling constants, Hz
13CNMRb
yield: %
Hl
HZ
JIZ
523
J16
J16'
C1
C2
70 65 58 55
3.41 3.38 3.38 3.38
3.00 3.00 3.02 3.01
9.3 9.1 9.3 9.3
9.3 9.1 9.3 9.3
10.3 10.2 10.2 10.1
4.6 4.6 4.6 4.6
75.38 75.48 75.55 75.56
81.52 79.69 79.82 79.81
'H NMR,b ppm
coupling constants, Hz
R, &,trans series yield," % HI HZ J1,Z 5 , n-CI5H31 65 3.94 3.28 3.0 OIsolated compound; yield calculated relative to the starting material.
523
J1'6
9.1
2.8c
13C NMRb Cl
J1'6'
CZ
5.2c 69.80 75.68 6 units relative to MelSi ( 6 = 0). 'Difficult to measure.
Table 11. Results of Open Epicutaneous Tests on Guinea Pigs
no. of animals reading with a test intensitv of 2 1 0.5 0 0 1 3 4 ~
sensitizer" 3c, R = n-C,oHzlb 3c, R = n-CloHzlb 3c, R = n-C1oH2iC 3c, R = n-C1oHZlc 3c, R = n-CIOHziC 3c, R = n-CIOHZIC 3d, R = n-C15H31b 3d, R = n-C15H31b 3d, R = n-C15H3lb 3d, R = n-C15H3iC 3d, R = n-C15H3iC 3d, R = n-C15H3iC 3d, R = n-C15H3lC 5 , R = n-C1,5H31' 5 , R = n-C15H3ib 5 , R = n-CijH31' 5 , R = n'C15H3lC 5 , R = n-C,,H,,' 7 (PDCIb
compd tested (wmol) 3, R = n-CloH21 (3.8) 3, R = n-CloHz1 (14) 3, R = n-CloHzl (3.8) 3, R = n-CloH21 (7.6) 3, R = n-CioH21 (14) 3, R = n-Ci5H31 (7.6) 3, R = n-Cl5H31 (3.8) 3, R = n-Ci5H31 (7.6) 7 (0.013) 3, R = n-Cl5H31 (3.8) 3, R = n-C15H31 (7.6) 3, R = n-CioH21 (7.6) 5, R = n-CijH31 (7.6) 5 , R = n-CIbH31 (3.8) 5 , R = n-Cl5H31 (7.6) 5 , R = n-C15H31 (3.8) 5 , R = n-C,hH31 (7.6) 3, R = n-CljH31 (7.6)
2
0 0 2 0 0 0 0
1 2
0 0 0 0 0 0 0 1 0
2 2 5 2 1 4 12 1 9 11 5
~~
2 4 1 3 6 8 2 6 4 2 1 1 5 6 4 2 1 2 0
0 0 1
2 5
av skin reactn 0.3 0.9 0.5 0.7 0.9 0.5 0.5 0.8 0.5 0.8 1.0 0.6 0.1 0.3 0.5 0.5 0.8 0.1 1.0 0
2 2 2 1 1 4 2 1 2 1 2 7 3 1 2 1 7 0 8
no. of Dositive animals 418 618 618 618 718
718 12/16 14/16 718 14/16 15/16 618 118 518 718 618 718 118 818 018
0 5 0 "No sensitization was obtained with 3-methyl- (3a, R = CH3) and 3-pentyl-1,2-cyclohexanediols(3b, R = n-C5Hll). In the two groups of eight guinea pigs used, no animal reacted. bSensitized by the FCAT method, using three injections (each other day) of a 1:1 FCA/saline
emulsion of the hapten.
7 (0.013) 3, R = n-C15H31 (7.6)
o;b- a;+ 01~
Sensitized by the FCAT method, using five injections.
Chemistry. The model chosen, as close as possible to 3-alkylpyrocatechols, was 3-alkyl-1,2-cyclohexanediols. The spatial arrangement that seemed closest to the o-diphenol appeared to be all-trans-3-alkyl-l,2-cyclohexanediols. To our knowledge, the only synthesis of pure all-trans-3-alkyl- (in this instance, 3-methyl) cyclohexanediols was described by Klein and Dunkelblum,15 their starting material being commercially available 3methyl-2-cyclohexenone. The same authors have described another synthesis of this compound from cyclohexenone16 unfortunately leading to a mixture of trans,trans and trans,cis isomers. We have now devised a synthesis based on the Michael addition of RzCuLi (or RMgX with CUI catalysis) to 2-cyclohexenone (1). In the first process, to a solution of R,CuLi (made in situ from RLi and CUI)in THF cooled at -60 "C was added 2-cyclohexenone. The resulting enolate was quenched with chlorotrimethylsilane and extracted. The crude [(trimethylsilyl)oxy]cyclohexene was then added to a solution of BH3-Me2S complex in THF.17 In the second process, R,CuLi was replaced by RMgBr (in the presence of CUI) (Scheme I). Only one stereomer (the all-trans derivative) was obtained in every instance, as evidenced by GC and NMR spectroscopy. The configuration was secured by the magnitudes of J12and JZ3,characteristic in cyclohexane (15) Klein, J.; Dunkelblum, E. Tetrahedron 1968, 24, 5701-5710. (16) Klein, J.; Levene, R.; Dunkelblum, E. Tetrahedron Lett. 1978, 2845-2848. (17) Brown, H. C.; Sharp, R. L. J . Am. Chem. Soc. 1968, 90, 2915-2927.
Scheme IIIa QH
a
4 a a =
5
6
RMgBr ( + CUI).
systems of a diaxial arrangement1* (Table I). Because of the presence of the bulky trimethylsiloxy (OTMS) group, diborane attack occurs exclusively from the nonhindered face (Scheme 11). All compounds, except for 3-methyl1,2-cyclohexanediol (3a), are new. Compared to the one-pot reaction of Michael addition/hydroboration of cyclohexenones, the yields are greatly improved. We have also prepared cis7truns-3-pentadecyl-1,2cyclohexanediol,which was needed (see discussion below) in order to eliminate one possible mechanism of sensitization to cyclohexanediols. The key material was cis2,3-epoxycyclohexano1. The classical way for the synthesis of this compound was direct epoxidation (m-chloroperbenzoic acid) of 2-cyclohexenol.1Q However, a 91 mixture of cisltrans isomer was obtained. In order to obtain di(18) Ziffer, H.; Seeman, J. I.; Highet, R. J.;Sokoloski, E. A. J. Org. Chem. 1974,39, 3698-3701. (19) Chavdarian, C. G.; Heathcock, C. H. Synth. Commun. 1976, 6, 277-280.
Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2 289 SENSlTILINL
I
TESTS
SLBSTILVLL:
2
(trans)
0 CONTROLS
K-C l U H 2
U
0 5
1 0
I 5
2 0
I ’
Average s k i n reaction
Figure 1. Compared skin sensitivity to trans,trans-n-decyl- and n-pentadecylcyclohexanediolsin guinea pigs.
rectly pure isomers without time-consuming purification (isomers with long hydrocarbon chains are difficult to separate), we used the Bongini et al. procedurez0as modified by Lipschutz,21namely, iodocarbonation of 2-cyclohexenol, followed by MeOH/K2C03treatment, leading to cis-2,3-epoxycyclohexanol(4) in 65% overall yield. Opening of cis-2,3-epoxycyclohexanolby RMgX (with CUI catalysis) led to cis,trans-3-alkyl-l,2-cyclohexanediol 5, which was easily purified by making cis-diol acetonide (Scheme 111).
Results and Discussion The results of experimental sensitization of guinea pigs to trans,trans-1,2-cyclohexanediolsappear in Table 11. The first striking result is the importance of the side-chain length as far as the sensitizing power is concerned: when the number of carbons was inferior to 10, no sensitization was obtained. Thus, 3-methyl- and 3-pentyl-1,2-cyclohexanediols were nonsensitizers. This was already known for 3-alkyl~atechols,~~ where an optimum 11-15 carbon chain was found. Importance of the 3-Alkyl Side Chain Length. None of the four 3-alkyl derivatives (3a-d) showed any skin toxicity or primary irritation effects. As far as the pentadecyl compound is concerned, this is in contrast with the high toxicity found for 3-alkylcatechols by Baer et al.23 In the cyclohexanediol series, only Clo and C15derivatives were able to induce ACD (allergic contact dermatitis). They, however, can be considered as weak sensitizers as compared to their catechols counterparts, a 300 times higher dose being required to elicit a positive skin test reaction. In order to make sure that 3-methyl- and 3pentylcyclohexanediols were nonsensitizers, a much higher dose was used in an attempt to elicit a positive reaction without result. Boosting the sensitization by two injections of alkylcyclohexanediol in FCA (Freund complete adjuvant, a suspension of killed mycobacteria in mineral oil) 1 week after the first test increased the sensitivity of the animals and the results were even more homogeneous (Table 11). Cross-sensitization reactions between Clo and C15diols (Figure 1) show results analogous to those with Baer’s corresponding catechols.zz Cross-Sensitization Reaction with Catechols. A striking result is the “one-way” cross-reaction between 3-PDC (or urushiol) sensitized animals and the 3-alkyl(20) Bongini, A.; Cardillo, G.; Orena, M.; Porzi, G.; Sandri, S. J. Org. Chem. 1982,47, 4626-4633. (21) Lipshutz, B. H.; Kozlowski, 3. A. J . Org. Chem. 1984, 49, 1147-1149. (22) Baer, H.; Watkins, R. C.; Kurtz, A. P.; Byck, J. S.; Dawson, C. R. J . Zmmunol. 1967, 99, 370-375. (23) Baer, H.; Watkins, R. C.; Kurtz, A. P.; Byck, J. S.; Dawson, C. R. J.Immunol. 1967, 99, 365-369.
I
u ,
0
I 5
I b
2 0
hueraic
\kin r r a c f i < n
Figure 2. Compared skin sensitivity to trans,trans-n-pentadecylcyclohexanediol and pentadecylcatechol (PDC). TESTS
SENSITIZING SUBSTANCE
2 Rm
2
(cis) C15H31
c]
CONTROLS
5;
(cis) R =
2
( t r a n s ) R = C15H,I
c
~
~
H
(trans)
R- ‘11H31
I 0
I
0.5
1.5
2.0
Average *kin reaction
Figure 3. Compared skin sensitivity to cis,trans- and trans,-
trans-n-pentadecylcyclohexanediols. Scheme IV. Possible Metabolites of trans, trans-3-Alkylcyclohexanediols
R
cyclohexanediols. Thus, cyclohexanediol-sensitized animals react to PDC and to urushiol, but the converse is not true, i.e. PDC- and urushiol-sensitized guinea pigs do not react to cyclohexanediols (Figure 2). Such one-way ~~ cross-reactions are known in contact d e r m a t i t i ~ . Strong allergens only generate a few clones. Clones of cross-reacting cells are therefore strong when weak-sensitizer animals are elicited with a strong hapten, as observed. “Lipophilic”or “Electrophilic”Mechanism? It is generally admitted that, in order to become an antigen, hapten binds to epidermal proteins with the formation of a covalent bond.8 In the case of 3-alkylcatechols, general belief is that in vivo oxidation of these derivatives into electrophilic o-quinones precedes nucleophilic attack (though SH or NH2 groups) by skin protein. The role of the side chain is, however, very important as shown by Dawson’s results.22 Catechols with long chains (Cll seems the optimum length) are stronger allergens. A possible explanation for this is that the chain is inserted into the cell membrane (possibly Langerhans cells) and the hydrophilic part emerges from it. In support for this hypothesis, double-headed haptensz5have been prepared in this laboratory and shown to induce ACD in guinea pigs: only the pyrocatechol “end” was recognized. (24) Sidi, E.; Hincky, J.; Hincky, M. Reu. Fr. Allerg. 1964, 1-19. (25) Marchand, B.; Benezra, C. J. Med. Chem. 1982,25, 650-653.
~
~
290 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2
The trans-cyclohexanediols could also be transformed in vivo into electrophiles: epoxide or diketone (Scheme IV). In order to eliminate the epoxide formation hywas pothesis, cis,trans-3-pentadecyl-l,2-cyclohexanediol prepared and used to sensitize a group of guinea pigs. The diol turned out to be a weak sensitizer and interestingly no cross-reaction was observed between the two groups of trans,trans and cis,trans sensitized guinea pigs (Figure 3). This excludes the possibility of a common metabolite (such as 3-pentadecyl-1,2-cyclohexanedione). At the same time, the epoxide route (impossible to be generated from a cis-diol) also seems excluded.
mixture was then processed as described for method A. trans,trans-3-Alkyl-l,%-cyclohexanediols 3. To a solution of BH3-Me2S (Aldrich) (150 mmol) in THF (75 mL, 2 M solution) was added dropwise crude 3-alkyl-1-[ (trimethylsilyl)oxy]cyclohexene (50 mmol) for 30 min. The mixture was stirred for 48 h, after which BH3-Me2S in excess was decomposed by slow addition of EtOH (50 mL). A 3 N NaOH aqueous solution (15 mL) was added, followed by dropwise addition of 30% H20z(15 mL), and the reaction mixture stirred for 2 h a t room temperature. Potassium carbonate was then added to saturation of the solution, the organic layer was separated, and aqueous layers were extracted several times with AcOEt. Combined organic layers were dried over MgS04 and concentrated by rotary evaporation to leave crude diol. Purification by "flash" chromatography gave pure 3-al-
kyl-l,2-cyclohexanediol. cis,trans-3-Alkyl-lf-cyclohexanediols 5. A stirred solution
7
Finally, the most important result from this study are probably the finding that nonelectrophilic, lipophilic derivatives are capable of inducing allergic contact dermatitis without the requirement of covalent bond formation. This seems to be the first true example of such a mechanism. The importance of lipophilicity in ACD mechanism has received confirmation at the immunological and cellular levels.26,27 Experimental Section General Methods. Proton NMR spectra were recorded on a Brucker 200-MHz spectrometer in CDCl, unless otherwise specified. 13CNMFt spectra were taken on the same spectrometer a t 50 MHz. Chemical shifts are reported in 6 with respect to CHC13as internal standard. Coupling constants (J)are expressed in hertz. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), m (multiplet). Infrared spectra were obtained on a Beckman Acculab spectrometer using CHC13solutions; peaks are reported in reciprocal centimeters. Melting points were determined on a Buchi Tottoli 510 apparatus and are uncorrected. GLC analyses were performed with a Girdel 300 gas chromatograph with a 5% OV-17 column. Dry solvents were freshly distilled before use. Tetrahydrofuran (THF) was distilled from sodium benzophenone. Triethylamine and hexamethylphosphoramide (HMPA) were distilled from powdered calcium hydride: all air- or moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. General Procedure for the Preparation of 3-Alkyl-l[(trimethylsilyl)oxy]-1-cyclohexene.Method A. To a cold (-40 "C) suspension of copper(1) iodide (14.3 g, 75 mmol) in dry THF (200 mL) was added RLi (150 mmol) in THF or Et20. The resulting solution was stirred a t -40 "C for 10 min, then cooled to -50 "C, and treated with 2-cyclohexen-1-one (4.81 mL, 50 mmol). After being stirred for 1h a t -40 to -50 "C, the mixture was cooled to -60 "C, and HMPA (8.74 mL, 50 mmol), triethylamine (27.9 mL, 200 mmol), and chloromethylsilane (25.4 mL, 200 mmol) were added. The mixture was allowed to warm to room temperature for over 1h. The reaction was poured into a saturated aqueous NH4Cl solution (200 mL) and extracted with hexane (3 X 100 mL). The combined extracts were dried (MgSO,) and concentrated under vacuum. Method B. A stirred solution of alkylmagnesium bromide (73 mmol) in T H F (200 mL) was treated with copper(1) iodide (1.43 g, 7.5 mmol) in one portion at room temperature and immediately cooled to -40 "C. The resulting mixture was treated with 2cyclohexen-1-one (4.81 mL, 50 mmol). After being stirred for 2 h a t -40 to -50 "C, the resulting enolate was quenched by consecutive addition of HMPA (8.74 mL, 50 mmol), EGN (13.93 mL, 100 mmol), and chlorotrimethylsilane (12.69 mL, 100 mmol). The (26) Darley, M. 0.;Post, W.; Hunter, R. L. J.Immunol. 1977, 118, 963-970. (27) Liischer, I. F. J. Med. Chem. 1984,27, 1502-1508.
of alkylmagnesium bromide (75 mmol) in T H F (200 mL) was treated with copper(1) iodide (1.43 g, 7.5 mmol) in one portion a t room temperature and immediately cooled to -40 "C. The resulting mixture was treated with cis-2,3-epoxycyclohexanolzo~21 (3.42 g, 30 mmol). After being stirred for 2 h at -40 "C, the mixture was allowed to warm to room temperature and stirred overnight in saturated aqueous NH4C1solution (200 mL) and extracted with hexane (3 x 100 mL). The combined extracts were dried (MgSO,) and concentrated under vacuum. The crude cis-diol (50 mmol) was treated overnight at room temperature with an excess of 2,2-dimethoxypropane (12.3 mL, 100 mmol) in acetone (100 mL) with a catalytic amount of p-TsOH. The resulting mixture was concentrated under vacuum and acetonide separated from 6 by flash chromatography (hexane/AcOEt, 91).$The crude acetonide was treated with MeOH (100 mL) and p-toluenesulfonic acid (catalytic amount) to release diol 5. After concentration under vacuum, purification by flash chromatography gave pure cis,trans-3-alkyl-l,2-cyclohexanediol 5. cis ,trans -3-Pentadecyl-1,2-cyclohexanediol ( 5 ) : white crystals; mp 68 "C; IR (CHCl,) 3580-3440 (P OH); 'H NMR (CDCI,) 6 0.88 (t, 3 H, J = 6.4 Hz), 1.26 (m, 29 H), 1.41-1.86 (m, 6 H), 3.28 (dd, 1 H, 5 2 3 = 9.1 Hz, J1t2 = 3.0 Hz), 3.94 (ddd, 1 H, 523 = 9.1 Hz, J1'6 2.8, J1qy 5.2 Hz); 13CNMR (CDCl3) 6 75.68, 69.80, 38.38,31.94,30.99, 30.04,29.70 (8C), 29.37 (SC),28.77, 26.73, 22.69, 19.23, 14.10. Anal. (C21H4202) C, H. 2-n -Pentadecyl-1,3-cyclohexanediol(6): IR (CHCl,) 3565-3420; 'H NMR (CDCl,) 6 0.88 (t,3 H, J = 6.4 Hz), 1.25 (m, 29 H), 1.46-1.78 (m, 2 H), 1.83-1.86 (m, 4 H), 3.73 (ddd, 2 H, J12 = 516 = 9.3 Hz, Jl, = 3.4 Hz). Anal. (C21H4202) C, H. 3-Methyl-1,2-cyclohexanediol(3a). Method A: white crystals;16mp 40 "C; IR (CHC13) 3610-3440; 'H NMR (CDCl,) 6 1.02 (m, 1H), 1.03 (d, 3 H, J = 6.4 Hz), 1.31 (m, 3 H), 1.67 (d, 2 H, J = 7.7 Hz), 1.96 (m, 1 H), 2.45 (s, 2 H), 2.95 (t-like dd, 1 H, 5 1 2 = 5 2 3 = 9.3 Hz), 341 (ddd, 1 H, J12 = 9.3 Hz, J 1 6 = 10.3 Hz, J16 = 4.6 Hz);13C NMR (CDC13) 6 81.52, 75.38, 37.72, 33.40, 33.03, 23.45, 18.11. 3-Pentyl-1,2-cyclohexanediol(3b). Method A: white crystals; mp 66 "C; IR (CHC13)3600-3440; 'H NMR (CDC1,) 6 0.88 (t, 3 H, J = 6.6 Hz), 1.27 (m, 9 H), 1.7 (m, 3 H), 1.95 (m, 2 H), 2.08 (m, 1 H), 3.00 (dd, appearing like a triplet, 1 H, J12 = 5 2 3 = 9.1 Hz), 3.38 (ddd, 1 H, 5 1 2 = 9.1 Hz, J16 = 10.2 Hz, J16' = 4.6 Hz); 13CNMR (CDCI,) 6 79.69,75.48,42.59,32.89,32.23,31.88, 29.96, 26.28, 23.41, 22.64, 14.00. Anal. (CllH220z) C, H. 3-Decyl-l,2-cyclohexanediol(3c):white crystals; mp 61 "C; IR (CHCl3) 3590-3420; 'H NMR (CDC13) 6 0.88 ( t , 3 H, J = 6.5 Hz), 1.26 (m, 19 H), 1.74 (m, 3 H), 1.97 (m, 2 H), 2.15 (m, 1 H), 3.02 (t-like dd, 1 H, J12= 5 2 3 = 9.3 Hz), 3.38 (ddd, 1 H, J12 = 9.3 Hz, J1,j = 10.2 Hz, JIG, = 4.6 Hz); 13CNMR (CDClJ 6 79.82,75.55, 42.60, 32.86, 31.93,30.07,29.96,29.67 (3C), 29.34 (ZC), 26.28,23.41, 22.67, 14.09. Anal. (C16H3202)c, H. trans ,trans-3-Pentadecyl-1f-cyclohexanediol(3d): white crystals; mp 72.5 "C; IR (CHC1,) 3600-3420; 'H NMR (CDClJ 6 0.88 (t, 3 H, J = 6.5 Hz), 1.26 (m, 29 H), 1.75 (m, 3 H), 1.95 (m, 2 H), 2.14 (m, 1 H), 3.01 (t-like dd, 1 H, J12= 5 2 3 = 9.3 Hz), 3.88 (ddd, 1 H, J12= 9.3 Hz, J16 = 10.1 Hz, J16, = 4.6 Hz); 13CNMR (CDC13)6 79.81, 75.06, 42.57, 32.89, 31.76, 30.05,30.02,29.67 (7c), 29.65, 29.32 (2C), 26.30, 23.43, 22.65, 14.10. Anal. (C21H4202) C, H. Biological Assays. Albino Himalayan spotted Fullingsdorf (from Hoffman La Roche, Basel) female guinea pigs weighing from
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J.Med. Chem. 1986,29, 291-295 300 to 500 g were sensitized as described by Klecak.28 On alternate days, the hapten, emulsified in Freund's complete adjuvant (FCA), was injected intradermally (0.1 mL) in the shaved nuchal region of the animal (in all, three injections, five after boost). The sensitizing molarity used was 0.12 M (FCA/saline (1:l)). After 15 days rest, the elicitation was conducted by an open epicutaneous test (OEt): 25 p L of an ethanol solution of diol (see Table (28) Klecak, G.; Geleick, H.; Frei, J. R. J. SOC.Cosmet. Chem. 1977, 28, 53-64.
29 1
I1 for amount of diols) was deposited on the shaved flank of the animal (on a 2-cm2surface using a standard circular stamp). Tests were read at the 48th h using the following scale: 0 = no reaction, 0.5 = slight erythema not covering the whole test area, 1 = erythema covering all the test area, 2 = erythema plus swelling of the test area, 3 = erythema plus swelling going well beyond the test area. Before any sensitization, irritation thresholds (primary toxicity) were determined on FCA-injected controls (same procedure as above for elicitation). Concentrations up to 5% in ethanol of cyclohexanediols were nontoxic. Control groups of eight animals (FCA treated) were used in each experiment.
Notes Pyrazolo[4,5-c ]quinolines. 2. Synthesis and Specific Inhibition of Benzodiazepine Receptor Binding Fabrizio Melani,f Lucia Cecchi,? Giovanna Palazzino,t Guido Filacchioni,*t Claudia Martini,$ Emanuela Pennacchi,t and Antonio Lucacchinit Dipartimento di Scienze Farmaceutiche, Universitd di Firenze, 50121 Firenze, and Istituto Policattedra di Discipline Biologiche, Universitd di Pisa, 56100 Pisa, Italy. Received April 26, 1985 A series of 1-aryl-3,5-dimethyl-4,5-dihydro-lH-pyrazolo[4,5-c]quinolin-4-ones (2a-e) and 1-aryl-3-methyl-1Hpyrazolo[4,5-c]quinolines(3-7a-e) bearing different substituents at position 4 were prepared and tested for their ability to displace specific [3H]flunitrazepam binding from bovine brain membranes. The 5-N-methyl derivatives 2a-c,e were the compounds that bound with the highest affinity within this class. The replacement of the carbonyl group with other substituents and the resulting aromatization of the pyridine moiety greatly decreased the binding affinity. From a Lineweaver-Burk analysis on the most active compound 2b, it appears that the inhibition is a competitive one.
Since the reports of Squires and Braestrupl and Mohler and Okada2 on the high affinity binding sites for benzodiazepines in rat brain tissues, a number of synthetic compounds with different structures have been found to possess high affinity for the benzodiazepine receptor either as agonists or as antag~nists.~ Non-benzodiazepine compounds with affinity for the benzodiazepine receptor could be of potential importance as tools for studying the receptor itself and eventually for the introduction into clinical use of new classes of compounds having the same properties of benzodiazepines. Cain et a1.4 in a study on 0-carbolines have reported some of the requirements that affect the affinity for the receptor. They stated that a planar heteroaromatic system containing at least one nitrogen atom is necessary and that a carbonyl group adjacent to the nitrogen atom greatly augments the binding to the receptor. Guzman et aL5recently verified the same requirements in other non-benzodiazepine compounds like canthines, isoquinolines, and imidazoquinolines. In the latter a phenyl substituent provides the necessary hydrophobicity for a better fitting. Following these findings we have prepared some pyrazoloquinolin-Conesbearing an aryl substituent at position 1or at position 2.6 A preliminary binding study on bovine brain membranes has shown that only the l-aryl-3methyl-4,5-dihydro-lH-pyrazolo[4,5-c]quinolin-4-ones possess activity in displacing specific [3H]flunitrazepam from its receptor site.6 Dipartimento di Scienze Farmaceutiche.
* Istituto Policattedra di Discipline Biologiche.
In order to establish structure-activity relationships for benzodiazepine receptor binding in this class of compounds, we are now reporting the synthesis and binding properties of some new l-arylpyrazolo[4,5-c]quinoline derivatives. Chemistry. l-Phenyl-3-methyl-4,5-dihydro-1" pyrazolo[4,5-c]quinolin-4-one(la),already reported in the literature,' was synthesized to compare its activity with that of the 1-aryl-3-methyl-4,5-dihydro-1H-pyrazolo[4,5c]quinolin-4-ones (lb-e) previously reported by US.^ Compounds la-e were reacted with methyl iodide to obtain the 5-methyl derivatives 2a-e. (1) Squire, R. F.; Braestrup, C. Nature (London) 1977,266,732. (2) Mohler, H.; Okada, T. Science 1977, 198, 849. (3) Lippa, A. S.; Critchet, D.; Sano, M. C.; Klepner, C. A.; Greenblatt, E. N.; Couplet, J.; Beer, B. Pharmacol. Biochem. Behau. 1979,10,831. Blanchard, J. C.; Boireau, A.; Garret, C.; Julau, L. Life Sci. 1978,24, 2417. Breastrup, C.; Nielsen, M.; Olsen, C. E. R o c . Natl. Acad. Sci. U.S.A. 1980, 77,2288. GuBrBmy, C. G. A.; Uzan, A. Life Sci. 1981, 28, 1439. Yokoyama, N.; Ritter, B.; Neubert, A. D. J. Med. Chem. 1982, 25, 337. (4) Cain, M.; Weber, R. W.; Guzman, F.; Cook, J. M.; Baker, S. A.; Rice, K. C.; Crawley, J. N.; Paul, S. M.; Skolnick, P. J . Med. Chem. 1982,25, 1081. (5) Guzman, F.; Cain, M.; Larscheid, P.; Hagen, T.; Cook, J. M.; Schweri, M.; Skolnick, P.; Paul, S. M. J . Med. Chem. 1984,27, 564. (6) Cecchi, L.; Melani, F.; Palazzino, G.; Filacchioni, G.; Martini, C.; Pennacchi, E.; Lucacchini, A. Farmaco, Ed. Sci. 1985,40, 509. (7) Knorr, L.; Jodicke, F. Chem. Ber. 1885,18,2256. Musierowicz, A.; Niementowski, S.; Tomasik, J. Rocz. Chem. 1928, 8, 325; Zentralblatt 1928,ZZ, 1882. Vul'fson, N. S.; Zhurin, R. B. Zh. Obsch. Khim. 1962, 32, 991.
0022-2623/86/1829-0291$01.50/0 0 1986 American Chemical Society
